Project Summary/Abstract Cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is a standard of care for the curative-intent treatment of muscle-invasive bladder cancer (MIBC). This project is based on the scientific premise that prospective molecular profiling can identify patients with MIBC (cT2-4aN0M0) for whom transurethral resection of the bladder tumor (TURBT) and systemic chemotherapy is curative without the need for RC. It builds upon prior retrospective data demonstrating that 1) deleterious alterations in DNA damage response (DDR) genes, most frequently in the nucleotide excision repair gene ERCC2, are predictive of response to cisplatin-based combination chemotherapy in MIBC and 2) small cohorts of patients who achieve clinical complete responses to NAC and refuse or are medically unfit for RC survive long-term with their bladders intact. The hypothesis that select patients with MIBC can be successfully managed with TURBT and chemotherapy alone, without the need for RC, will be tested in Aim 1 in the context of a prospective, multicenter clinical trial performed by the Alliance for Clinical Trials in Oncology (A031701). In this study, pre- treatment diagnostic TURBT samples will undergo next-generation sequencing analysis of 468 cancer- associated genes. All patients will receive dose-dense gemcitabine and cisplatin for 6 cycles followed by clinical re-staging and will be managed with either bladder preservation or RC based upon 1) post- chemotherapy response as assessed by repeat cystoscopy, TURBT, and imaging, and 2) somatic mutation status of DDR-related genes in the patient's pre-treatment tumor. Patients with deleterious somatic alterations in at least 1 of 9 DDR-associated genes who achieve a clinical complete response or down-staging to noninvasive disease (<cT1) following NAC will be candidates for bladder preservation. Patients with DDR gene mutations who have ?cT1 disease after chemotherapy will undergo RC, as will all DDR gene wild-type patients, regardless of response. Recognizing that ~60% of NAC responders lack somatic DDR gene alterations, whole exome sequencing will be performed in Aim 2, using the tumor material collected pre- treatment from all patients enrolled on A031701, to identify additional biomarkers of chemo-sensitivity, including mutation signatures of DDR deficiency. In Aim 3, we will assess whether analysis of cell-free DNA from blood and urine is a sensitive, noninvasive method to identify patients with minimal residual disease and to explore tumor heterogeneity and its relationship to drug resistance and disease recurrence. The proposed studies rely extensively on support from the Biospecimen Repository and the Biostatistics & Bioinformatics Core to achieve the project's translational objectives. If successful, the studies proposed could significantly expand the use of organ-sparing therapy for the curative-intent treatment of patients with MIBC. The prospective molecular characterization platform used in this project could also accelerate testing of novel immunotherapy or targeted approaches in patients unlikely to respond to cisplatin-based NAC.